Table S4. Differential expression analysis of the transcriptome of WT and ΔpldA strains from The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri

Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support PldA, which is widespread across Gram-negative bacteria, as an important factor for the bacterial life cycle, particularly in human pathogens.

外膜磷脂酶A（Outer membrane phospholipase A，OMPLA）是一类定位于革兰氏阴性细菌外膜的酶类。该酶具有广泛的底物特异性，其部分底物定位于细胞包膜。通常而言，该酶的催化活性仅能通过多种方法扰动细胞包膜完整性来诱导激活。尽管外膜磷脂酶A作为膜蛋白在大肠杆菌（Escherichia coli）中已被深入研究，且在诸多其他细菌性致病菌中呈组成型表达，但目前对于其在细菌感染过程中的功能仍知之甚少。本研究中，蛋白质组学与转录组学数据显示，福氏志贺菌（Shigella flexneri）中的外膜磷脂酶A被命名为PldA，其在常规培养条件下既可稳定细菌细胞膜，又参与细菌感染过程。一系列生理学实验证实，ΔpldA突变菌株的细菌外膜与周质空间出现结构紊乱。此外，ΔpldA突变菌株的III型分泌系统（type III secretion system）表达水平下调，导致其在宿主细胞中的内化效率降低。本研究结果证实，广泛存在于革兰氏阴性细菌中的PldA是细菌生命周期中的重要调控因子，对于人类致病菌而言尤为如此。